- Email: [email protected]
Histological analysis of somatic embryogenesis and organogenesis induced from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.)
Plant Science 161 (2001) 415– 421 www.elsevier.com/locate/plantsci
Histological analysis of somatic embryogenesis and organogenesis induced from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) K. Chengalrayan a, S. Hazra b, M. Gallo-Meagher a,* b
a Agronomy Department, Uni6ersity of Florida, Gaines6ille, FL 32611 -0300, USA Plant Tissue Culture Di6ision, National Chemical Laboratory, Pashan Road, Pune 411008, India
Received 6 December 2000; received in revised form 26 March 2001; accepted 9 April 2001
Abstract The initiation and development of somatic embryos and organogenic buds in peanut (Arachis hypogaea L.) obtained from mature zygotic embryo-derived leaflets (MZELs) was studied histologically. The MZELs were cultured on Murashige and Skoog (MS) basal medium supplemented with 20 mg l − 1 2,4-dichlorophenoxyacetic acid (2,4-D) for embryogenic mass induction. Somatic embryos developed from these masses following transfer to a medium containing 3 mg l − 1 2,4-D and were germinated on hormone-free medium. A combination of 4 mg l − 1 a-naphthaleneacetic acid (NAA) and 5 mg l − 1 6-benzylamino purine (BAP) was optimum to induce organogenic buds which developed into shoots once placed on a medium containing 0.5 mg l − 1 BAP and 0.5 mg l − 1 kinetin. Histological studies of explants at various developmental stages of somatic embryogenesis and organogenesis revealed that both somatic embryos and organogenic buds developed directly from the mesophyll layers of MZELs, and both were of multicellular origin. Initially, MZELs underwent periclinal division when cultured on MS medium containing 20 mg l − 1 2,4-D giving rise to somatic embryos. Interestingly, MZELs underwent anticlinal division when cultured on MS medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP that resulted in organogenesis. Initial failure of somatic embryos to convert into plantlets was due to malformation of the shoot meristem. However, transfer to medium containing 2 mg l − 1 BAP and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ) resulted in the appearance of broad shoot apices which could be induced to produce plantlets. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arachis hypogaea L.; Histology; Mature zygotic embryo-derived leaflets; Organogenesis; Somatic embryogenesis
1. Introduction In vitro regeneration via somatic embryogenesis or organogenesis is important for clonal propagation and is usually an integral part of genetic transformation studies. Since the discovery of plant growth regulators and their use in plant tissue culture, significant progress has been made in in vitro regeneration. Studies aimed at understanding morphogenetic differentiation have resulted in voluminous literature describing the various factors that influence morphogenic responses in plant tissues [1]. Although advances are being made toward
* Corresponding author. Tel.: + 1-352-3921823x206; fax: +1-3523927248. E-mail address: [email protected] (M. Gallo-Meagher).
better understanding the metabolic processes correlated with regeneration [2,3], determining conditions for in vitro plant regeneration is still largely an empirical process. As a result, in vitro regeneration can be difficult to achieve for some plant species or particular genotypes within a species. Although extensive research has been carried out on in vitro embryogenesis and organogenesis in cultivated peanut (Arachis hypogaea L.) [4–8], there is little information on the development of somatic embryos and shoots or their cellular origins. A report on somatic embryogenesis in peanut from immature zygotic embryos describes the process as direct without intervening callus [9]. Gill and Saxena [10] also observed that somatic embryogenesis resulting from seedling explants was direct, and initial stages of somatic embryogenesis were described histologically. Most peanut somatic em-
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 3 9 - 3
416
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
bryos fail to develop into plantlets with a 0–25% conversion frequency being reported [4,11– 14]. Recently, Little et al. [8] reported that depending upon the genotype used, 20–60% of somatic embryos could be converted into plantlets. Similar problems exist for Arachis pintoi where only 5– 10% of the somatic embryos were converted into plantlets [15]. However, a higher conversion percentage, 86–92%, from initially abnormal peanut somatic embryos could be achieved upon transfer to medium containing 2 mg l − 1 6-benzylamino purine (BAP) and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ) [16]. There are only a few reports in which different stages of somatic embryogenesis and organogenesis induced from the same explant source have been histologically examined [15,17–20]. In sunflower, Bronner et al. [19] showed that both morphogenetic events were multicellular in origin and occurred directly without intervening callus. The epidermis and hypocotyl cortex were the origins of morphogenesis and initial cell divisions were periclinal. Histological analysis of organogenesis induced in immature tissues of Stylosanthes scabra revealed that neoformed buds developed from deep-seated vascular nodule structures derived from callus tissue [18]. Examination of somatic embryogenesis in this species also suggested that its response was direct and of multicellular origin [18]. In wheat, both somatic embryogenesis and shoot organogenesis were observed in the same callus tissue that contained typical stages of somatic embryoid development, and there was evidence for de novo shoot formation [20]. More recently both developmental pathways, somatic embryogenesis and oraganogenesis, were induced in A. pintoi leaf explants via an initial callus phase [15]. Further information regarding developmental stages of embryogenesis and organogenesis are needed to study the developmental pattern of in vitro morphogenesis in cultivated peanut. Such work may lead to a better understanding of in vitro development in peanut, and consequently result in higher regeneration rates that should benefit clonal propagation and transformation work. This paper describes at the cellular level the developmental pattern of peanut embryogenesis and organogenesis induced from the same explant source. 2. Methods
2.1. Regeneration Embryo axes were excised from cotyledons of the peanut (Arachis hypogaea L.) cultivar J.L. 24, a Spanish market type. Axes were surface sterilized for 3 min with 0.1% mercuric chloride (HgCl2). Excess HgCl2 was removed by three to five washings with sterile, distilled water and the axes were soaked in water for 12–16 h prior to removal of folded, zygotic embryo-derived
leaflets (MZELs) (Fig. 1A). Thirty MZELs were cultured per Petri dish (55×15 mm). Methods described earlier were followed for the induction of somatic embryos [4,16] and organogenic buds [21]. Briefly, to induce embryogenesis, MZELs were cultured for 4 weeks on Murashige and Skoog (MS) [22] basal salts and vitamins supplemented with 6% (w/v) sucrose and 20 mg l − 1 2,4-dichlorophenoxyacetic acid (2,4-D). Explants were then transferred to MS medium containing 6% (w/v) sucrose and 3 mg l − 1 2,4-D for the development of somatic embryos. After 4 weeks of culture, embryos were germinated on MS basal medium and then for conversion were transferred to medium containing MS salts and vitamins with 2 mg l − 1 6-benzylamino purine (BAP) and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ). For shoot bud induction, leaflets were cultured on MS basal salts and vitamins supplemented with 2% (w/v) sucrose, 4 mg l − 1 a-naphthaleneacetic acid (NAA) and 5 mg l − 1 BAP for 4 weeks. Explants were then transferred to the same basal medium containing 0.5 mg l − 1 BAP and 0.5 mg l − 1 kinetin for shoot development. The pH of all media was adjusted to 5.8 and media were solidified with 0.7% (w/v) agar. Cultures were incubated at 259 2°C under white fluorescent light at 32 mM m − 2 s − 1 with a 16 h photoperiod.
2.2. Histology To study the developmental stages of somatic embryogenesis, MZELs were fixed in FAA (formaldehyde: acetic acid: ethanol, 5:5:90) for 48 h at day 0 and after day 3, 7, 10, 15, 30, 60 and 120 of culture on somatic embryo induction medium. For organogenesis, MZELs were fixed at day 0 and after day 4, 7, 15, 21 and 30 of culture, on organogenesis induction medium. Histological studies were carried out according to standard procedures with tissues fixed in FAA solution for 48 h being passed through a t-butanol dehydration series and embedded in paraffin wax at 58°C [23]. Sections 8 mm in thickness were cut on a rotatory microtome (Reichert–Jung 2050) and fixed on glass slides. Sections were de-waxed with xylene for 5– 10 min and then double stained with haematoxyline and eosin by passing the slides through an ethanol series. Sections were mounted in DPX-4 mountant (BDH Industries Ltd., India) before microscopic examination (Docuval, Carl Zeiss, Germany).
3. Results and discussion
3.1. Somatic embryogenesis At the time of culture initiation, cross sections of MZELs showed a single-layered epidermis and four
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
417
Fig. 1. Mature zygotic embryo-derived leaflets (MZELs) at different time intervals on MS medium supplemented with 6% (w/v) sucrose and 20 mg l − 1 2,4-D for somatic embryo induction. (A) Folded MZEL dissected from an embryo axis (X13). (B) Cross section of MZEL, initial explant showing four mesophyll layers surrounded by epidermis. Cells are filled with storage reserves (X250). (C) Cross section showing changes in the cells of a 3 day cultured explant. Arrows ( ) in epidermis and mesophyll cells indicate that these cells are undergoing anticlinal and periclinal division, respectively, (X400). (D) Cross section showing numerous periclinal divisions in the mesophyll layers after 7 days in culture. Granules were not apparent ( ) in these dividing cells, but could be observed in the static cells (X250). (E) Cross section showing several layers of cells resulting from continuous periclinal divisions after 10 days in culture to form a small bulge (X250). (F) Cross section of an explant showing a large bulge after 15 days of culture. This was the result of both anticlinal and periclinal divisions (X64). Granules were not apparent in any of these cells. (G) Embryogenic mass development on either side of the midrib of the MZEL after 30 days in culture. (H) Bulges appearing on either side of the original midrib after 30 days in culture and (I) clearly showing disintegration of the remaining mother explant tissue (X32). (J) Reappearance of granules ( ) within cells after 30 days of culture (X250).
418
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
layers of subepidermis (mesophyll cells) (Fig. 1B). All cells were filled with granules that could be lipid, protein and/or starch bodies since the leaflet explants were isolated from embryo axes. Ultrastructural analysis of mesophyll cells (predivision embryogenic cells) from Dactylis glomerata showed that they were filled with both starch granules and lipid bodies [24]. Earlier, it was shown that 2 weeks of culturing MZELs in the presence of 20 mg l − 1 2,4-D resulted in the induction of a pair of embryogenic masses forming on either side of the midrib of these leaflet explants [4,21,25]. Cross sections passing through MZELs that were cultured for only 3 days on somatic embryogenesis induction medium revealed a proliferation of mesophyll cells and the presence of an intact epidermis (Fig. 1C). Anticlinal divisions on the adaxial side of the epidermis, as well as periclinal divisions in the mesophyll cell layers of MZELs were observed (Fig. 1C). At this early stage of embryo induction, divisions of the mesophyll cells were asynchronous. Cell divisions appeared restricted to regions close to the midrib at the base of the explant. The granular structures noted at the time of culture initiation disappeared in the dividing cells. In an independent study involving biochemical estimations, it was observed that storage lipids present in these leaflet explants are depleted during the initial stages of embryogenesis [26]. On day 7 of embryogenesis induction, most of the mesophyll cells of the MZELs were now undergoing periclinal division (Fig. 1D) that resulted in the formation of a small bulge that could be easily observed on the explants. However, at this culture stage some cells were in a static phase and as a result, granules still could be observed in these cells. Periclinal divisions continued following 10 days of culture without disruption of the epidermal layer (Fig. 1E). After 15 days of culture, both periclinal and anticlinal divisions of the mesophyll layers were observed, and these cells were characterized by a dense cytoplasm and nuclei containing enlarged nucleoli (Fig. 1F). By this time, all cells had divided resulting in the formation of a large bulge, and granules were no longer apparent in any of the cells. After 30 days of culture, cells formed globular structures on either side of what was the MZEL midrib (Fig. 1G, H). At this culture stage, what remained of the mother explant began to deteriorate (Fig. 1I), and granules reappeared in the cells (Fig. 1J). It is at this stage that peanut somatic embryos have been shown to accumulate lipids [26]. Therefore, these granules are most likely lipid bodies. When these globular explants were transferred to medium containing 3 mg l − 1 2,4-D, they continued to divide by periclinal divisions giving rise to meristematic zones (Fig. 2A, B). Occasionally, cotyledonary stage embryos were observed (Fig. 2C). In a portion of the
cotyledonary stage embryos, shoot poles were flattened (Fig. 2D). This type of meristem has been earlier described as a disk meristem [27] that fails to give rise to shoots. The shoot apex was broad in the somatic embryos and shoot development could not be achieved when transferred to germination medium. In our earlier study [4], it was presumed that the failure of somatic embryos to form plantlets was either due to malformation of the apex or due to insufficient storage products in the less developed cotyledons. However, increasing storage products such as triglycerides within these somatic embryos, also failed to convert them into plantlets [28]. The present histological studies suggest that malformation of the apex is most likely responsible for the lack of shoot regeneration. By manipulating the culture conditions, the structure and behavior of these somatic embryos could be altered [29]. Therefore, when these embryos were transferred to medium containing 2 mg l − 1 BAP plus 3 mg l − 1 kinetin, or 5 mg l − 1 TDZ, multiple shoots (Fig. 2E) appeared from broad shoot apices (Fig. 2F). These results clearly show that peanut somatic embryos did not originate from single cells, but from multicellular, mesophyll regions that initially undergo a series of periclinal divisions. The epidermal layer participated by means of anticlinal divisions and developed into the epidermis of the new shoots.
3.2. Organogenesis Culturing MZELs on organogenic induction medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP resulted in direct formation of adventitious shoot buds at the base of the explants, i.e. from the same region where embryogenesis was induced. Cross sections representing different developmental stages are presented in Fig. 3. During the initial 4 days of culture, cells of the MZELs enlarged. On day 7 of organogenic induction, mesophyll cells underwent anticlinal division giving rise to cell clusters throughout the mesophyll layers (Fig. 3A). At this culture stage, the anticlinal divisions were strictly limited to the mesophyll cells. Subsequently, periclinal and anticlinal divisions occurred within these newly formed cell clusters. Cell divisions in the epidermal layer were regularly anticlinal. From the cell clusters, meristematic buds appeared (Fig. 3B) in 15 days. Fig. 3C, D shows well-developed buds after 21 and 30 days of culture, respectively. Fig. 3D is a typical illustration of young primordia that eventually formed adventitious shoots. It is clear that the meristems originated from several cells and that mesophyll cell layers were involved in adventitious shoot formation. The epidermal cells underwent anticlinal divisions and as such formed the epidermis of the new adventitious shoots.
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
Our earlier studies, along with this report, have shown that MZELs of cultivar J.L. 24 are excellent explants for both the induction of somatic embryos and organogenic buds [4,21,25]. Genes that trigger cellular differentiation in culture can be selectively influenced by growth regulators [30,31]. That is the case for peanut where the mesophyll layers of the MZELs responded differently in vitro depending upon the growth regulator treatments that led to somatic embryogenesis or organogenesis. To our knowledge, such a predictable site of induction has not been reported earlier. The histological observations reported here provide evidence that both embryogenesis and organogenesis were direct, i.e. without an intermediate callus phase. The induction of morphogenetic events was surprisingly rapid in the peanut MZELs. Morphological changes were visible after 7 days of culture, while histological sections displayed cellular modifications as early as 3–4 days post culture. Both the somatic embryos and shoots described here were produced by simultaneous divisions of several cells and thus had multicellular origins. This histological work has been extended to include another variety, SunOleic 97R [32]. Similar results, including the timing and type of cellular divisions leading to somatic embryogenesis and organogenesis have been observed
419
in this Runner market type peanut (data not shown). In conclusion, depending on the growth regulators employed, somatic embryogenesis or organogenesis could be induced from the same peanut explant source. Histological examination of early events during embryogenesis and organogenic bud formation from peanut MZELs indicated that both morphogenetic pathways were initiated from the mesophyll layers and were multicellular in origin. Embryogenesis began with periclinal cell divisions of the mesophyll cells in contrast to organogenesis that began with anticlinal divisions of these cells. Subsequently, both embryos and shoot buds developed by both periclinal and anticlinal divisions. This system may prove useful for future studies elucidating the genes involved in periclinal and anticlinal divisions in vitro, as well as those genes that influence the subsequent differentiation processes of embryogenesis and organogenesis in peanut.
Acknowledgements The authors thank Dr K.H. Quesenberry for critical review of the manuscript. This work was approved for publication as Journal Series No. R-07894 by the Flor-
Fig. 2. Somatic embryos at different time intervals on MS medium supplemented with 6% (w/v) sucrose and 3 mg l − 1 2,4-D. (A) Somatic embryos developed from embryogenic masses after 60 days in culture. (B) Longitudinal section of a somatic embryo after 60 days of culture. The cells continued to divide periclinally, and meristematic zones appeared (X200). (C) Cotyledonary embryo without a shoot meristem (X25.6). (D) Longitudinal section of a cotyledonary embryo showing a flat apex and a poorly developed apical meristem (X80). (E) Germinated, abnormal somatic embryos with shoots induced in the presence of 5 mg l − 1 TDZ at 120 days of culture. Plantlets show multiple shoots with small leaves. (F) Longitudinal section showing multiple shoot meristems that developed from the apex in the presence of TDZ (X64).
420
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
Fig. 3. Mature zygotic embryo-derived leaflets (MZELs) at different time intervals in MS medium supplemented with 2% (w/v) sucrose, 4 mg l − 1 NAA and 5 mg l − 1 BAP for induction of organogenesis. (A) Anticlinal divisions in mesophyll cells of an MZEL after 7 days of culture (X450). (B) Section of 15-day-old explant showing initiation of organogenic buds (X250). (C) Organogenic response in MZELs after 21 days in basal medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP. (D) Section of a differentiated organogenic bud showing leaf initials after 30 days in culture (X100).
ida Agricultural Experiment Station. We also express appreciation to the Council of Scientific and Industrial Research (CSIR), India for granting a fellowship (1992–1997) to K. Chengalrayan.
References [1] K.M.T.T. Van, Control of morphogenesis in in vitro cultures, Ann. Rev. Plant Physiol. 32 (1981) 291 –311. [2] P. Lambe´ , H.S.N. Mutambel, J-G. Fouche´ , R. Deltour, J-M. Foidart, T. Gaspar, DNA methylation as a key in regulation of organogenic totipotency and plant neoplastic progression, In Vitro Cell. Dev. Biol. 33P (1997) 155 – 162. [3] J. Cairney, N. Xu, J. Mackay, J. Pullman, Special symposium: in vitro plant recalcitrance transcript profiling: a tool to assess the development of confer embryos, In Vitro Cell. Dev. Biol. 36P (2000) 155 – 162. [4] K. Chengalrayan, S.S. Sathey, S. Hazra, Somatic embryogenesis from mature embryo-derived leaflets, Plant Cell Rep. 13 (1994) 578 – 581. [5] R. Gill, P. Ozias-Akins, Thidiazuron induced highly morphogenetic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants, In Vitro Cell. Dev. Biol. 35P (1999) 445 – 450. [6] Y. Akasaka, H. Daimon, M. Mii, Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron, Plant Sci. 156 (2000) 169 – 175.
[7] P. Lakshmanan, A. Taji, Somatic embryogenesis in leguminous plants, Plant Biol. 2 (2000) 136 – 148. [8] E.L. Little, Z.V. Maganua, W.A. Parrott, A protocol for repetitive somatic embryogenesis from mature peanut epicotyls, Plant Cell Rep. 19 (2000) 351 – 357. [9] S. Hazra, S.S. Sathey, A.F. Mascarenhas, Direct somatic embryogenesis of peanut (Arachis hypogaea L), BioTechnology 7 (1989) 217 – 218. [10] R. Gill, P.K. Saxena, Direct somatic embryogenesis and regeneration of plants from seedling explants of peanut (Arachis hypogaea): promotive role of thidiazuron, Can. J. Bot. 70 (1992) 1186 – 1192. [11] P. Ozias-Akins, Plant-regeneration from immature embryos of peanut, Plant Cell Rep. 8 (1989) 217 – 218. [12] H.Y. Wetzstein, C.M. Baker, The relationship between somatic embryo morphology and conversion in peanut (Arachis hypogaea L.), Plant Sci. 91 (1993) 81 – 89. [13] S. Eapen, L. George, Plant-regeneration from leaf-disks of peanut and pigeonpea — influence of benzyladenine, indoleacetic-acid and indoleacetic-acid amino-acid conjugates, Plant Cell Tissue Org. Cult. 35 (1993) 223 – 227. [14] R.L. Reddy, G.M. Reddy, Factors affecting direct somatic embryogenesis and plant regeneration in groundnut, Arachis hypogaea L, Indian J. Exp. Biol. 31 (1993) 57 – 60. [15] H.Y. Rey, A.M. Scocchi, A.M. Gonzalez, L.A. Mroginski, Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture, Plant Cell Rep. 19 (2000) 856 – 862. [16] K. Chengalrayan, V.B. Mhaske, S. Hazra, High frequency conversion of abnormal peanut somatic embryos, Plant Cell Rep. 16 (1997) 783 – 786.
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421 [17] M.M. Altamura, A. Cersosimo, C. Majoli, M. Crespan, Histological study of embryogenesis and organogenesis from anthers of Vitis rupestris du Lot cultured in vitro, Protoplasma 171 (1992) 134 – 141. [18] M.C. Dornelas, M.L.C. Vieira, B. Appezzato-Da-Gloria, Histological analysis of organogenesis induced in immature tissues of Stylosanthes scabra, Ann. Bot. 70 (1992) 477 –482. [19] R. Bronner, G. Jeannin, G. Hahne, Early cellular events during organogenesis and somatic embryogenesis induced on immature zygotic embryos of sunflower (Helianthus-annuus), Can. J. Bot. 72 (1994) 239 – 248. [20] S.L. Kothari, A. Varshney, Morphogenesis in long-term maintained immature embryo-derived callus of wheat (Triticum aesti6um L.) — histological evidence for both somatic embryogenesis and organogenesis, J. Plant Biochem. Biotechnol. 7 (1998) 93 – 98. [21] K. Chengalrayan, V.B. Mhaske, S. Hazra, In vitro regulation of morphogenesis in peanut (Arachis hypogaea L.), Plant Sci. 110 (1995) 259 – 269. [22] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473 – 497. [23] A.K. Sharma, A. Sharma, Chromosome Techniques; Theory and Practice, Butterworths, London, 1980, pp. 71 –80. [24] A. Vasilenko, J.K. McDaniel, B.V. Conger, Ultrastructural anal-
.
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
421
ysis of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata, In Vitro Cell. Dev. Biol. 36P (2000) 51 – 56. K. Chengalrayan, V.B. Mhaske, S. Hazra, Genotypic control of peanut somatic embryogenesis, Plant Cell Rep. 17 (1998) 522 – 525. V.B. Mhaske, S. Hazra, Appearance of storage lipids (triglycerides) in somatic embryos of peanut (Arachis-hypogaea L), In Vitro Cell. Dev. Biol. 30P (1994) 113 – 116. P.V. Ammirato, in: R.R. Henke, K.W. Hughes, M.P. Constantin, A. Hollaender (Eds.), Tissue Culture in Forestry and Agriculture, New York, Plenum, 1985, pp. 9 – 29. V.B. Mhaske, K. Chengalrayan, S. Hazra, Influence of osmotica and abscisic acid on triglyceride accumulation in peanut somatic embryos, Plant Cell Rep. 17 (1998) 742 – 746. P.V. Ammirato, The regulation of somatic embryo development in plant cell cultures: suspension culture techniques and hormone requirements, BioTechnology 1 (1983) 68 – 74. T.A. Thorpe, Organogenesis in vitro: structural, physiological and biochemical aspects, Int. Rev. Cyst. Suppl. 11A (1980) 71 – 111. T.A. Thorpe, B. Stefania, Regulation of plant organogenesis, Adv. Cell. Cult. 1 (1981) 213 – 239. D.W. Gorbet, Registration of ‘SunOleic 95R’ peanut, Crop Sci. 31 (1997) 1392.
Histological analysis of somatic embryogenesis and organogenesis induced from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) K. Chengalrayan a, S. Hazra b, M. Gallo-Meagher a,* b
a Agronomy Department, Uni6ersity of Florida, Gaines6ille, FL 32611 -0300, USA Plant Tissue Culture Di6ision, National Chemical Laboratory, Pashan Road, Pune 411008, India
Received 6 December 2000; received in revised form 26 March 2001; accepted 9 April 2001
Abstract The initiation and development of somatic embryos and organogenic buds in peanut (Arachis hypogaea L.) obtained from mature zygotic embryo-derived leaflets (MZELs) was studied histologically. The MZELs were cultured on Murashige and Skoog (MS) basal medium supplemented with 20 mg l − 1 2,4-dichlorophenoxyacetic acid (2,4-D) for embryogenic mass induction. Somatic embryos developed from these masses following transfer to a medium containing 3 mg l − 1 2,4-D and were germinated on hormone-free medium. A combination of 4 mg l − 1 a-naphthaleneacetic acid (NAA) and 5 mg l − 1 6-benzylamino purine (BAP) was optimum to induce organogenic buds which developed into shoots once placed on a medium containing 0.5 mg l − 1 BAP and 0.5 mg l − 1 kinetin. Histological studies of explants at various developmental stages of somatic embryogenesis and organogenesis revealed that both somatic embryos and organogenic buds developed directly from the mesophyll layers of MZELs, and both were of multicellular origin. Initially, MZELs underwent periclinal division when cultured on MS medium containing 20 mg l − 1 2,4-D giving rise to somatic embryos. Interestingly, MZELs underwent anticlinal division when cultured on MS medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP that resulted in organogenesis. Initial failure of somatic embryos to convert into plantlets was due to malformation of the shoot meristem. However, transfer to medium containing 2 mg l − 1 BAP and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ) resulted in the appearance of broad shoot apices which could be induced to produce plantlets. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arachis hypogaea L.; Histology; Mature zygotic embryo-derived leaflets; Organogenesis; Somatic embryogenesis
1. Introduction In vitro regeneration via somatic embryogenesis or organogenesis is important for clonal propagation and is usually an integral part of genetic transformation studies. Since the discovery of plant growth regulators and their use in plant tissue culture, significant progress has been made in in vitro regeneration. Studies aimed at understanding morphogenetic differentiation have resulted in voluminous literature describing the various factors that influence morphogenic responses in plant tissues [1]. Although advances are being made toward
* Corresponding author. Tel.: + 1-352-3921823x206; fax: +1-3523927248. E-mail address: [email protected] (M. Gallo-Meagher).
better understanding the metabolic processes correlated with regeneration [2,3], determining conditions for in vitro plant regeneration is still largely an empirical process. As a result, in vitro regeneration can be difficult to achieve for some plant species or particular genotypes within a species. Although extensive research has been carried out on in vitro embryogenesis and organogenesis in cultivated peanut (Arachis hypogaea L.) [4–8], there is little information on the development of somatic embryos and shoots or their cellular origins. A report on somatic embryogenesis in peanut from immature zygotic embryos describes the process as direct without intervening callus [9]. Gill and Saxena [10] also observed that somatic embryogenesis resulting from seedling explants was direct, and initial stages of somatic embryogenesis were described histologically. Most peanut somatic em-
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 3 9 - 3
416
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
bryos fail to develop into plantlets with a 0–25% conversion frequency being reported [4,11– 14]. Recently, Little et al. [8] reported that depending upon the genotype used, 20–60% of somatic embryos could be converted into plantlets. Similar problems exist for Arachis pintoi where only 5– 10% of the somatic embryos were converted into plantlets [15]. However, a higher conversion percentage, 86–92%, from initially abnormal peanut somatic embryos could be achieved upon transfer to medium containing 2 mg l − 1 6-benzylamino purine (BAP) and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ) [16]. There are only a few reports in which different stages of somatic embryogenesis and organogenesis induced from the same explant source have been histologically examined [15,17–20]. In sunflower, Bronner et al. [19] showed that both morphogenetic events were multicellular in origin and occurred directly without intervening callus. The epidermis and hypocotyl cortex were the origins of morphogenesis and initial cell divisions were periclinal. Histological analysis of organogenesis induced in immature tissues of Stylosanthes scabra revealed that neoformed buds developed from deep-seated vascular nodule structures derived from callus tissue [18]. Examination of somatic embryogenesis in this species also suggested that its response was direct and of multicellular origin [18]. In wheat, both somatic embryogenesis and shoot organogenesis were observed in the same callus tissue that contained typical stages of somatic embryoid development, and there was evidence for de novo shoot formation [20]. More recently both developmental pathways, somatic embryogenesis and oraganogenesis, were induced in A. pintoi leaf explants via an initial callus phase [15]. Further information regarding developmental stages of embryogenesis and organogenesis are needed to study the developmental pattern of in vitro morphogenesis in cultivated peanut. Such work may lead to a better understanding of in vitro development in peanut, and consequently result in higher regeneration rates that should benefit clonal propagation and transformation work. This paper describes at the cellular level the developmental pattern of peanut embryogenesis and organogenesis induced from the same explant source. 2. Methods
2.1. Regeneration Embryo axes were excised from cotyledons of the peanut (Arachis hypogaea L.) cultivar J.L. 24, a Spanish market type. Axes were surface sterilized for 3 min with 0.1% mercuric chloride (HgCl2). Excess HgCl2 was removed by three to five washings with sterile, distilled water and the axes were soaked in water for 12–16 h prior to removal of folded, zygotic embryo-derived
leaflets (MZELs) (Fig. 1A). Thirty MZELs were cultured per Petri dish (55×15 mm). Methods described earlier were followed for the induction of somatic embryos [4,16] and organogenic buds [21]. Briefly, to induce embryogenesis, MZELs were cultured for 4 weeks on Murashige and Skoog (MS) [22] basal salts and vitamins supplemented with 6% (w/v) sucrose and 20 mg l − 1 2,4-dichlorophenoxyacetic acid (2,4-D). Explants were then transferred to MS medium containing 6% (w/v) sucrose and 3 mg l − 1 2,4-D for the development of somatic embryos. After 4 weeks of culture, embryos were germinated on MS basal medium and then for conversion were transferred to medium containing MS salts and vitamins with 2 mg l − 1 6-benzylamino purine (BAP) and 3 mg l − 1 kinetin, or 5 mg l − 1 thidiazuron (TDZ). For shoot bud induction, leaflets were cultured on MS basal salts and vitamins supplemented with 2% (w/v) sucrose, 4 mg l − 1 a-naphthaleneacetic acid (NAA) and 5 mg l − 1 BAP for 4 weeks. Explants were then transferred to the same basal medium containing 0.5 mg l − 1 BAP and 0.5 mg l − 1 kinetin for shoot development. The pH of all media was adjusted to 5.8 and media were solidified with 0.7% (w/v) agar. Cultures were incubated at 259 2°C under white fluorescent light at 32 mM m − 2 s − 1 with a 16 h photoperiod.
2.2. Histology To study the developmental stages of somatic embryogenesis, MZELs were fixed in FAA (formaldehyde: acetic acid: ethanol, 5:5:90) for 48 h at day 0 and after day 3, 7, 10, 15, 30, 60 and 120 of culture on somatic embryo induction medium. For organogenesis, MZELs were fixed at day 0 and after day 4, 7, 15, 21 and 30 of culture, on organogenesis induction medium. Histological studies were carried out according to standard procedures with tissues fixed in FAA solution for 48 h being passed through a t-butanol dehydration series and embedded in paraffin wax at 58°C [23]. Sections 8 mm in thickness were cut on a rotatory microtome (Reichert–Jung 2050) and fixed on glass slides. Sections were de-waxed with xylene for 5– 10 min and then double stained with haematoxyline and eosin by passing the slides through an ethanol series. Sections were mounted in DPX-4 mountant (BDH Industries Ltd., India) before microscopic examination (Docuval, Carl Zeiss, Germany).
3. Results and discussion
3.1. Somatic embryogenesis At the time of culture initiation, cross sections of MZELs showed a single-layered epidermis and four
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
417
Fig. 1. Mature zygotic embryo-derived leaflets (MZELs) at different time intervals on MS medium supplemented with 6% (w/v) sucrose and 20 mg l − 1 2,4-D for somatic embryo induction. (A) Folded MZEL dissected from an embryo axis (X13). (B) Cross section of MZEL, initial explant showing four mesophyll layers surrounded by epidermis. Cells are filled with storage reserves (X250). (C) Cross section showing changes in the cells of a 3 day cultured explant. Arrows ( ) in epidermis and mesophyll cells indicate that these cells are undergoing anticlinal and periclinal division, respectively, (X400). (D) Cross section showing numerous periclinal divisions in the mesophyll layers after 7 days in culture. Granules were not apparent ( ) in these dividing cells, but could be observed in the static cells (X250). (E) Cross section showing several layers of cells resulting from continuous periclinal divisions after 10 days in culture to form a small bulge (X250). (F) Cross section of an explant showing a large bulge after 15 days of culture. This was the result of both anticlinal and periclinal divisions (X64). Granules were not apparent in any of these cells. (G) Embryogenic mass development on either side of the midrib of the MZEL after 30 days in culture. (H) Bulges appearing on either side of the original midrib after 30 days in culture and (I) clearly showing disintegration of the remaining mother explant tissue (X32). (J) Reappearance of granules ( ) within cells after 30 days of culture (X250).
418
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
layers of subepidermis (mesophyll cells) (Fig. 1B). All cells were filled with granules that could be lipid, protein and/or starch bodies since the leaflet explants were isolated from embryo axes. Ultrastructural analysis of mesophyll cells (predivision embryogenic cells) from Dactylis glomerata showed that they were filled with both starch granules and lipid bodies [24]. Earlier, it was shown that 2 weeks of culturing MZELs in the presence of 20 mg l − 1 2,4-D resulted in the induction of a pair of embryogenic masses forming on either side of the midrib of these leaflet explants [4,21,25]. Cross sections passing through MZELs that were cultured for only 3 days on somatic embryogenesis induction medium revealed a proliferation of mesophyll cells and the presence of an intact epidermis (Fig. 1C). Anticlinal divisions on the adaxial side of the epidermis, as well as periclinal divisions in the mesophyll cell layers of MZELs were observed (Fig. 1C). At this early stage of embryo induction, divisions of the mesophyll cells were asynchronous. Cell divisions appeared restricted to regions close to the midrib at the base of the explant. The granular structures noted at the time of culture initiation disappeared in the dividing cells. In an independent study involving biochemical estimations, it was observed that storage lipids present in these leaflet explants are depleted during the initial stages of embryogenesis [26]. On day 7 of embryogenesis induction, most of the mesophyll cells of the MZELs were now undergoing periclinal division (Fig. 1D) that resulted in the formation of a small bulge that could be easily observed on the explants. However, at this culture stage some cells were in a static phase and as a result, granules still could be observed in these cells. Periclinal divisions continued following 10 days of culture without disruption of the epidermal layer (Fig. 1E). After 15 days of culture, both periclinal and anticlinal divisions of the mesophyll layers were observed, and these cells were characterized by a dense cytoplasm and nuclei containing enlarged nucleoli (Fig. 1F). By this time, all cells had divided resulting in the formation of a large bulge, and granules were no longer apparent in any of the cells. After 30 days of culture, cells formed globular structures on either side of what was the MZEL midrib (Fig. 1G, H). At this culture stage, what remained of the mother explant began to deteriorate (Fig. 1I), and granules reappeared in the cells (Fig. 1J). It is at this stage that peanut somatic embryos have been shown to accumulate lipids [26]. Therefore, these granules are most likely lipid bodies. When these globular explants were transferred to medium containing 3 mg l − 1 2,4-D, they continued to divide by periclinal divisions giving rise to meristematic zones (Fig. 2A, B). Occasionally, cotyledonary stage embryos were observed (Fig. 2C). In a portion of the
cotyledonary stage embryos, shoot poles were flattened (Fig. 2D). This type of meristem has been earlier described as a disk meristem [27] that fails to give rise to shoots. The shoot apex was broad in the somatic embryos and shoot development could not be achieved when transferred to germination medium. In our earlier study [4], it was presumed that the failure of somatic embryos to form plantlets was either due to malformation of the apex or due to insufficient storage products in the less developed cotyledons. However, increasing storage products such as triglycerides within these somatic embryos, also failed to convert them into plantlets [28]. The present histological studies suggest that malformation of the apex is most likely responsible for the lack of shoot regeneration. By manipulating the culture conditions, the structure and behavior of these somatic embryos could be altered [29]. Therefore, when these embryos were transferred to medium containing 2 mg l − 1 BAP plus 3 mg l − 1 kinetin, or 5 mg l − 1 TDZ, multiple shoots (Fig. 2E) appeared from broad shoot apices (Fig. 2F). These results clearly show that peanut somatic embryos did not originate from single cells, but from multicellular, mesophyll regions that initially undergo a series of periclinal divisions. The epidermal layer participated by means of anticlinal divisions and developed into the epidermis of the new shoots.
3.2. Organogenesis Culturing MZELs on organogenic induction medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP resulted in direct formation of adventitious shoot buds at the base of the explants, i.e. from the same region where embryogenesis was induced. Cross sections representing different developmental stages are presented in Fig. 3. During the initial 4 days of culture, cells of the MZELs enlarged. On day 7 of organogenic induction, mesophyll cells underwent anticlinal division giving rise to cell clusters throughout the mesophyll layers (Fig. 3A). At this culture stage, the anticlinal divisions were strictly limited to the mesophyll cells. Subsequently, periclinal and anticlinal divisions occurred within these newly formed cell clusters. Cell divisions in the epidermal layer were regularly anticlinal. From the cell clusters, meristematic buds appeared (Fig. 3B) in 15 days. Fig. 3C, D shows well-developed buds after 21 and 30 days of culture, respectively. Fig. 3D is a typical illustration of young primordia that eventually formed adventitious shoots. It is clear that the meristems originated from several cells and that mesophyll cell layers were involved in adventitious shoot formation. The epidermal cells underwent anticlinal divisions and as such formed the epidermis of the new adventitious shoots.
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
Our earlier studies, along with this report, have shown that MZELs of cultivar J.L. 24 are excellent explants for both the induction of somatic embryos and organogenic buds [4,21,25]. Genes that trigger cellular differentiation in culture can be selectively influenced by growth regulators [30,31]. That is the case for peanut where the mesophyll layers of the MZELs responded differently in vitro depending upon the growth regulator treatments that led to somatic embryogenesis or organogenesis. To our knowledge, such a predictable site of induction has not been reported earlier. The histological observations reported here provide evidence that both embryogenesis and organogenesis were direct, i.e. without an intermediate callus phase. The induction of morphogenetic events was surprisingly rapid in the peanut MZELs. Morphological changes were visible after 7 days of culture, while histological sections displayed cellular modifications as early as 3–4 days post culture. Both the somatic embryos and shoots described here were produced by simultaneous divisions of several cells and thus had multicellular origins. This histological work has been extended to include another variety, SunOleic 97R [32]. Similar results, including the timing and type of cellular divisions leading to somatic embryogenesis and organogenesis have been observed
419
in this Runner market type peanut (data not shown). In conclusion, depending on the growth regulators employed, somatic embryogenesis or organogenesis could be induced from the same peanut explant source. Histological examination of early events during embryogenesis and organogenic bud formation from peanut MZELs indicated that both morphogenetic pathways were initiated from the mesophyll layers and were multicellular in origin. Embryogenesis began with periclinal cell divisions of the mesophyll cells in contrast to organogenesis that began with anticlinal divisions of these cells. Subsequently, both embryos and shoot buds developed by both periclinal and anticlinal divisions. This system may prove useful for future studies elucidating the genes involved in periclinal and anticlinal divisions in vitro, as well as those genes that influence the subsequent differentiation processes of embryogenesis and organogenesis in peanut.
Acknowledgements The authors thank Dr K.H. Quesenberry for critical review of the manuscript. This work was approved for publication as Journal Series No. R-07894 by the Flor-
Fig. 2. Somatic embryos at different time intervals on MS medium supplemented with 6% (w/v) sucrose and 3 mg l − 1 2,4-D. (A) Somatic embryos developed from embryogenic masses after 60 days in culture. (B) Longitudinal section of a somatic embryo after 60 days of culture. The cells continued to divide periclinally, and meristematic zones appeared (X200). (C) Cotyledonary embryo without a shoot meristem (X25.6). (D) Longitudinal section of a cotyledonary embryo showing a flat apex and a poorly developed apical meristem (X80). (E) Germinated, abnormal somatic embryos with shoots induced in the presence of 5 mg l − 1 TDZ at 120 days of culture. Plantlets show multiple shoots with small leaves. (F) Longitudinal section showing multiple shoot meristems that developed from the apex in the presence of TDZ (X64).
420
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421
Fig. 3. Mature zygotic embryo-derived leaflets (MZELs) at different time intervals in MS medium supplemented with 2% (w/v) sucrose, 4 mg l − 1 NAA and 5 mg l − 1 BAP for induction of organogenesis. (A) Anticlinal divisions in mesophyll cells of an MZEL after 7 days of culture (X450). (B) Section of 15-day-old explant showing initiation of organogenic buds (X250). (C) Organogenic response in MZELs after 21 days in basal medium containing 4 mg l − 1 NAA and 5 mg l − 1 BAP. (D) Section of a differentiated organogenic bud showing leaf initials after 30 days in culture (X100).
ida Agricultural Experiment Station. We also express appreciation to the Council of Scientific and Industrial Research (CSIR), India for granting a fellowship (1992–1997) to K. Chengalrayan.
References [1] K.M.T.T. Van, Control of morphogenesis in in vitro cultures, Ann. Rev. Plant Physiol. 32 (1981) 291 –311. [2] P. Lambe´ , H.S.N. Mutambel, J-G. Fouche´ , R. Deltour, J-M. Foidart, T. Gaspar, DNA methylation as a key in regulation of organogenic totipotency and plant neoplastic progression, In Vitro Cell. Dev. Biol. 33P (1997) 155 – 162. [3] J. Cairney, N. Xu, J. Mackay, J. Pullman, Special symposium: in vitro plant recalcitrance transcript profiling: a tool to assess the development of confer embryos, In Vitro Cell. Dev. Biol. 36P (2000) 155 – 162. [4] K. Chengalrayan, S.S. Sathey, S. Hazra, Somatic embryogenesis from mature embryo-derived leaflets, Plant Cell Rep. 13 (1994) 578 – 581. [5] R. Gill, P. Ozias-Akins, Thidiazuron induced highly morphogenetic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants, In Vitro Cell. Dev. Biol. 35P (1999) 445 – 450. [6] Y. Akasaka, H. Daimon, M. Mii, Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron, Plant Sci. 156 (2000) 169 – 175.
[7] P. Lakshmanan, A. Taji, Somatic embryogenesis in leguminous plants, Plant Biol. 2 (2000) 136 – 148. [8] E.L. Little, Z.V. Maganua, W.A. Parrott, A protocol for repetitive somatic embryogenesis from mature peanut epicotyls, Plant Cell Rep. 19 (2000) 351 – 357. [9] S. Hazra, S.S. Sathey, A.F. Mascarenhas, Direct somatic embryogenesis of peanut (Arachis hypogaea L), BioTechnology 7 (1989) 217 – 218. [10] R. Gill, P.K. Saxena, Direct somatic embryogenesis and regeneration of plants from seedling explants of peanut (Arachis hypogaea): promotive role of thidiazuron, Can. J. Bot. 70 (1992) 1186 – 1192. [11] P. Ozias-Akins, Plant-regeneration from immature embryos of peanut, Plant Cell Rep. 8 (1989) 217 – 218. [12] H.Y. Wetzstein, C.M. Baker, The relationship between somatic embryo morphology and conversion in peanut (Arachis hypogaea L.), Plant Sci. 91 (1993) 81 – 89. [13] S. Eapen, L. George, Plant-regeneration from leaf-disks of peanut and pigeonpea — influence of benzyladenine, indoleacetic-acid and indoleacetic-acid amino-acid conjugates, Plant Cell Tissue Org. Cult. 35 (1993) 223 – 227. [14] R.L. Reddy, G.M. Reddy, Factors affecting direct somatic embryogenesis and plant regeneration in groundnut, Arachis hypogaea L, Indian J. Exp. Biol. 31 (1993) 57 – 60. [15] H.Y. Rey, A.M. Scocchi, A.M. Gonzalez, L.A. Mroginski, Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture, Plant Cell Rep. 19 (2000) 856 – 862. [16] K. Chengalrayan, V.B. Mhaske, S. Hazra, High frequency conversion of abnormal peanut somatic embryos, Plant Cell Rep. 16 (1997) 783 – 786.
K. Chengalrayan et al. / Plant Science 161 (2001) 415–421 [17] M.M. Altamura, A. Cersosimo, C. Majoli, M. Crespan, Histological study of embryogenesis and organogenesis from anthers of Vitis rupestris du Lot cultured in vitro, Protoplasma 171 (1992) 134 – 141. [18] M.C. Dornelas, M.L.C. Vieira, B. Appezzato-Da-Gloria, Histological analysis of organogenesis induced in immature tissues of Stylosanthes scabra, Ann. Bot. 70 (1992) 477 –482. [19] R. Bronner, G. Jeannin, G. Hahne, Early cellular events during organogenesis and somatic embryogenesis induced on immature zygotic embryos of sunflower (Helianthus-annuus), Can. J. Bot. 72 (1994) 239 – 248. [20] S.L. Kothari, A. Varshney, Morphogenesis in long-term maintained immature embryo-derived callus of wheat (Triticum aesti6um L.) — histological evidence for both somatic embryogenesis and organogenesis, J. Plant Biochem. Biotechnol. 7 (1998) 93 – 98. [21] K. Chengalrayan, V.B. Mhaske, S. Hazra, In vitro regulation of morphogenesis in peanut (Arachis hypogaea L.), Plant Sci. 110 (1995) 259 – 269. [22] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473 – 497. [23] A.K. Sharma, A. Sharma, Chromosome Techniques; Theory and Practice, Butterworths, London, 1980, pp. 71 –80. [24] A. Vasilenko, J.K. McDaniel, B.V. Conger, Ultrastructural anal-
.
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
421
ysis of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata, In Vitro Cell. Dev. Biol. 36P (2000) 51 – 56. K. Chengalrayan, V.B. Mhaske, S. Hazra, Genotypic control of peanut somatic embryogenesis, Plant Cell Rep. 17 (1998) 522 – 525. V.B. Mhaske, S. Hazra, Appearance of storage lipids (triglycerides) in somatic embryos of peanut (Arachis-hypogaea L), In Vitro Cell. Dev. Biol. 30P (1994) 113 – 116. P.V. Ammirato, in: R.R. Henke, K.W. Hughes, M.P. Constantin, A. Hollaender (Eds.), Tissue Culture in Forestry and Agriculture, New York, Plenum, 1985, pp. 9 – 29. V.B. Mhaske, K. Chengalrayan, S. Hazra, Influence of osmotica and abscisic acid on triglyceride accumulation in peanut somatic embryos, Plant Cell Rep. 17 (1998) 742 – 746. P.V. Ammirato, The regulation of somatic embryo development in plant cell cultures: suspension culture techniques and hormone requirements, BioTechnology 1 (1983) 68 – 74. T.A. Thorpe, Organogenesis in vitro: structural, physiological and biochemical aspects, Int. Rev. Cyst. Suppl. 11A (1980) 71 – 111. T.A. Thorpe, B. Stefania, Regulation of plant organogenesis, Adv. Cell. Cult. 1 (1981) 213 – 239. D.W. Gorbet, Registration of ‘SunOleic 95R’ peanut, Crop Sci. 31 (1997) 1392.